Safe and effective gene transfer techniques for gene therapy have been studied for a long time, resulting in development of various gene transfer vehicles and gene delivery systems. In particular, vectors based on adenoviruses and retroviruses, and nonviral vectors using liposomes, cationic lipids and cationic polymers have been developed as gene transfer vehicles. However, there are significant problems when viruses are used as vehicles for transfer of therapeutic genes into target cells, as follows. The transferred genes can negatively affect function of host genes after integration into host chromosome, and there is no evidence for the transferred gene do not activate oncogenes. In addition, if viral genes are continuously expressed even at a small amount, autoimmune response may be induced. Moreover, if a variant of the virus used as a gene transfer vehicle emerges in a host, the host may become infected with the variant, and the host immune system may not effectively protect itself from the variant. For these reasons, rather than the vectors based on viruses, gene delivery systems using liposomes, or cationic lipids or polymers are preferred, and related studies aim to improve the alternative systems. Such nonviral gene transfer vectors are less effective than the viral vectors, but are advantageous in terms of safety due to mild side effects and being economical due to low cost production, thereby allowing industrial production of improved nonviral vectors.
A micelle is a single layer sphere, which is spontaneously formed by self-assembly of molecules having both hydrophilic and hydrophobic groups in an aqueous environment to maximize thermodynamic stability. Block copolymers are able to form self-assembled micelles in an aqueous solution. The inside of the micelles is hydrophobic and thus can easily entrap water-insoluble drugs, and the surface of the micelles is hydrophilic and thus facilitates solubilization of the water-insoluble drugs, thereby allowing its use for drug delivery. Micelles having the hydrophobic core and the hydrophilic shell are stabilized in an aqueous environment by hydrophobic interaction, or ionic interaction between polyelectrolytes having opposite charges. A polyethylene glycol (PEG)-conjugated polyelectrolyte spontaneously associates with another polyelectrolyte having an opposite charge to form complexes having a micellar structure, which are called polyelectrolyte complex micelles (Kataoka, K., Togawa, H., Harada, A., Yasugi, K., Matsumoto, T., Katayoshe, S., Macromolecules. 29, 8556-8557, 1996). The polyelectrolyte complex micelles are more attractive than other drug delivery systems, such as microspheres or nanoparticles, due to their properties of having a very small size and a very uniform size distribution, and being a self-associated structure, thereby facilitating quality control and reproduction of pharmaceutical preparations.
Polymers used for drug delivery to a target site of the body should be biocompatible. A representative example of such biocompatible polymers is polyethylene glycol (PEG). PEG, which has gained approval for in vivo use from the Food and Drug Administration (FDA) of USA, has been utilized for a long time in a broad range of applications from improvement of protein characteristics and surface modification of polymers used in drug delivery systems to gene transfer. PEG, which is one of the most widely used biocompatible polymers, has excellent water solubility, and low toxicity and immunogenicity. In addition, PEG can strongly inhibit absorption of proteins to the polymers used in drug delivery by modifying the surface properties of the polymers.
On the other hand, oligonucleotides have been used in treating diseases in humans and animals. For example, some antisense oligonucleotides are known to regulate expression of genes related to viral and fungal infections and metabolic diseases. Typically, antisense means a complementary oligonucleotide to a target nucleic acid sequence, where the oligonucleotide can hybridize to the target sequence. When a target gene is determined, a nucleic acid sequence sufficiently complementary, which is specifically hybridizing to a part of the target gene, is selected to accomplish desired inhibition of the target gene. However, antisense oligonucleotides are problematic with respect to their delivery to target cells, their lifetime in target cells, and their delivery efficiency to target cells through the plasma membrane. Since the backbone of the linear oligonucleotide is composed of repeating sugar and phosphate residues held together by phosphodiester bonds, most oligonucleotides are easily degraded in cells, especially by nuclease attack. In addition, owing to their short half-life of about 20 min, oligonucleotides should be continuously delivered to target cells at a proper concentration to achieve their therapeutic effect. Oligonucleases sensitive to nuclease digestion can have improved stability by introduction of phosphothioate groups (Milligan, J. F., Mateucci, M. D., Martin, J. C., J. Med. Chem. 36, 1923-1937, 1993), or 2-O-allyl groups (Fisher, T. L., Terhorst, T., Cao, X., Wagner, R. W., Nucleic Acid Res. 21, 3857-3865, 1993). However, techniques related to effective penetration of oligonucleotides through plasma membrane are still under development.
At present, antisense oligonucleotides are delivered to target cells by microinjection, a gene transfer system using cationic polymers or lipids, or a method of directly dispersing oligonucleotides in culture media However, other methods except for the microinjection method do not have high efficiency in delivery of antisense oligonucleotides to target cells.